Outline

z Fed-batch operation
• Models
• Advantages
• Disadvantages
• Example: penicillin production
z Perfusion culture
• Advantages
• Disadvantages
Fed-Batch Operation
z In fed-batch culture, nutrients are
continuously or semi-continuously added to
a system, while effluent is removed
discontinuously
• Usually used to overcome substrate inhibition or
catabolite repression
z Recall that in batch culture the concentration
of biomass at a certain time is given by:
X = X0 +Y M
X /S (S0 − S ) (9.27)
 Schematic of a Fed-Batch Culture
1) 2)

3)
Fed-Batch Culture
z When biomass concentration in a fed-batch
reactor reaches its maximum (Xm), the substrate
concentration is very low (S<<S0), and also
X>>X0, therefore:
X m = YX S S 0
M

z If, for a fed batch reactor containing some cells

(inoculum), a nutrient feed is started at a flow
rate of F, with a substrate concentration of S0,
the total amount of biomass in the vessel is
Xt=VX, where V is the culture volume at time t,
and Xt is the biomass at time t
Fed-Batch Culture
z The rate of increase of culture volume in a fed
batch reactor is: dV
=F (9.28)
dt
z Integrating:
V = V0 + Ft (9.29)

z The biomass concentration in the vessel at any

time is:
X=X Vt
(9.30)
Fed-Batch Culture
z The rate of change in biomass concentration is:

( )
dX V dX t dt − X t (dV dt )
= (9.31)
2
dt V

z Since dXt/dt=µnetXt, dV/dt=F, and F/V=D, eq.

9.31 becomes:
dX
= (µ net − D )X (9.32)
dt
Fed-Batch Culture
z When the substrate is totally consumed, S≈0,
and X=Xm
• At this point, dX/dt=0, and the system is at quasi-steady
state (nutrient consumption rate is nearly equal to
nutrient feed rate), therefore:
µ net = D (9.33)

z If maintenance energy can be neglected,

S Ks D
µ net = µm and S≅ (9.34)
Ks + S µm − D
Fed-Batch Culture
z The balance on the rate-limiting substrate
without maintenance energy is:
dS t
µ net X t
(9.36)
= FS 0 − M
dt YX S
• Where St is the total amount of the rate-limiting
substrate in the culture, and S0 is the concentration of S
in the feedstream
• At quasi-steady state, Xt=VXm, and essentially all
substrate is consumed, therefore:
dX t  dV 
= Xm  = X m F = FYX S S 0
M
(9.37)
dt  dt 
Fed-Batch Culture
z Integration of eq. 9.37 from t=0 to t, with the
initial biomass concentration in the reactor
being X 0t yields:
X = X + FY
t t
0
M
X S S 0t (9.38)

• That is, the total amount of cells in the culture

increases linearly with time
• Dilution rate and µnet decrease with time
• Since µnet=D at quasi-steady state, the growth rate
is controlled by the dilution factor
Fed-Batch Culture
z For product formation in a fed-batch reactor, at
quasi-steady state (S<<S0): P ≅ YP S S 0 (9.41)
z Or the potential product output is:
t
dP
= qp X t (9.42)
dt
z When the specific rate of product formation (qp)
is constant:
FP ≈ YP S S 0 F (9.43)

• Where Pt is the total amount of product in culture

Fed-Batch Culture
z Substituting Xt=(V0+Ft)Xm into eq. 9.41 yields:
 Ft 
P = P + q p X m V0 + t
t
0
t
(9.42)
 2 
z Integration of eq. 9.42 yields:
t
dP
= q p X m (V0 + Ft ) (9.43)
dt
z Eq. 9.43 can be written in terms of product:
V0  V0 Dt 
P = P0 + q p X m  + t (9.44)
V V 2 
Fed-Batch Culture at Quasi-
Steady State
(a) Variation of culture
volume, V, specific growth
rate, µ, cell, X, and
substrate, S, concentration
with time at quasi-steady
state
(b) Variation of product
concentration, P, with time
at quasi-steady state in a
single cycle of a fed-batch
culture
(a)
Example 9.3
Solution
Advantages of Fed-Batch Culture
z Production of high cell densities due to extension of
working time (particularly important in the production of
growth-associated products)
z Controlled conditions for the provision of substrates
during the fermentation
z Control over the production of by-products, or catabolite
repression effects, due to limited provision of only those
substrates solely required for product formation
z Allows the replacement of water lost via evaporation
z Alternative mode of operation for fermentations involving
bioremediation of toxic substrates (cells can only
metabolize a certain quantity at a time), or low solubility
compounds
z No additional special pieces of equipment are required to
convert from batch to fed-batch operation
Disadvantages of Fed-Batch Culture
z Requires previous analysis of the microorganism, its
requirements, and an understanding of its physiology
with respect to productivity
z Requires a substantial amount of operator skill for
set-up, definition and development of the process
z In a cyclic fed-batch culture, care must be taken in
the design of the process to ensure that toxins do not
accumulate to inhibitory levels, and that nutrients
other than those incorporated into the feed medium
do not become limiting
• Also, if many cycles are run, the accumulation of non-
producing or low-producing mutants may result
Fed Batch Culture: Penicillin
Production
z Fermentation is divided in two phases - the rapid-growth
phase during which the culture grows at the maximum
specific growth rate, and the slow-growth phase in which
penicillin is produced
z During the rapid-growth phase:
• An excess of glucose causes an accumulation of acids, and a
biomass oxygen demand greater than the aeration capacity of
the fermentor
• Glucose starvation may result in the organic nitrogen in the
medium being used as a carbon source, resulting in a high pH
and inadequate biomass formation
z During the production (slow-growth) phase:
• Feed rates should be designed to limit the growth rate and
oxygen consumption, such that a high rate of penicillin
synthesis is achieved, and sufficient dissolved oxygen is
available in the medium
Perfusion Culture
z Perfusion systems are most often used for
animal cell culture
z The basic characteristics are constant medium
flow, cell retention, and, in some cases,
selective removal of dead cells
z Cell retention is usually achieved by
membranes or screens, or by a centrifuge
capable of selective cell removal
• When a membrane is used, the system has some
characteristics of an immobilized cell system
Schematic of a Perfusion System
Advantages of Perfusion Culture
z Potential removal of cellular debris and
inhibitory by-products
z Removal of enzymes (e.g. proteases) released
by dead cells that may destroy or damage
product
z Shorter exposure time of product to potentially
harsh production conditions (e.g. high or low
pH)
z High per-unit volumetric productivity due to
high cell density and metabolism
z Essentially constant environment
Disadvantages of Perfusion Culture
z A large amount of medium is typically used
• This is expensive, due to the high costs of raw
materials for the medium, in addition to the costs of
preparing and sterilizing the medium
z Nutrients in the medium are less completely
utilized than in batch or fed-batch systems
z Costs for waste treatment increase
z Must consider the trade-off of improved
product quality and reactor productivity with the
associated increased expense